1. Immunoassay Technology
A number of techniques are known and have been described for the measurement of small quantities of biological materials. Of these techniques, the area of immunoassays has been extensively reviewed and the technique forms the basis of many commercially available assay kits.
For more than thirty years, immunoassay has been the method of choice for measuring low analyte concentrations in complex biological fluids. The procedure is equally applicable to the measurement of low molecular weight compounds such as drugs, steroids and the like, as well as large molecular weight compounds such as protein molecules. The technique combines sensitivity and specificity. Immunoassays are used in basic biological research to investigate the physiological and possible pathological role of a wide range of potent biologically active substances, including cyclic nucleotides, prostglandins, leukotrienes, growth factors, steroid hormones and cytokines. Such research often leads to the identification of new therapeutic agents. Immunoassays are often used in the pharmaceutical industry in many aspects of drug development processes. These range from drug screening, toxicological, pharmacological and pharmacokinetic studies, through to clinical trials. Immunoassays have had their greatest impact in the area of clinical diagnostic tests. The technique has been employed for many years in hospital clinical biochemistry laboratories to diagnose disease and metabolic disorders. The technique was introduced in 1959 by Berson & Yalow. (Yalow, R. S. and Berson S. A., Assay of plasma insulin in human subjects by immunologic methods, Nature, (1959), 184, 1684). The combination of a signal which could be detected and a protein molecule (an antibody) which binds specifically and avidly to the analyte of interest lies at the heart of all immunoassay procedures. Assay designs have proliferated over the last thirty years, as have the different types of signal reagents and detection systems. Sophisticated instruments with associated computer hardware have been developed with the aim of increasing sample throughput. Further background information relating to immunoassay techniques can be found in ‘The Immunoassay Handbook, (Wild, D. G. Ed, Stockton Press, New York, (1994), which deals with many of the concepts associated with immunoassay technology which are pertinent to the present invention. It considers, for example, competitive (also termed ‘labelled analyte’ or ‘limited reagent’) and immunometric (‘labelled antibody’ or ‘reagent excess’) systems.
The earliest methods were those which involved a step of separating the bound analyte from the free, in order to be able to measure the amount of bound analyte. Various separation methods have been described, including charcoal absorption, ammonium sulphate precipitation, magnetic particles (‘Amerlex™’), etc. More recently, solid supports have been utilised for immunoassay procedures, including the walls of microtitre well plates.
A more recent development has been the introduction of homogeneous radioimmunoassay technology, notably the technique of scintillation proximity assays (SPA) covered by U.S. Pat. No. 4,568,649. Scintillation proximity assay is a radioisotopic assay technique which has gained wide acceptance in recent years, and is applicable to radioimmunoassays, as well as to radio-receptor and enzyme assays. The technique relies on the observation that β-particles emitted from radioisotopes will travel only a limited distance in an aqueous environment (in the case of tritium β-particles, this is 1.5 μm), before the energy is dissipated. In SPA, the target of interest is immobilised to a small to microsphere containing scintillant. When a radioisotopically labelled molecule is brought into close proximity with the microsphere, O-particle energy is transferred effectively to the scintillant, thereby causing the emission of light. Labelled molecules which remain free in solution are undetected because they are too distant from the scintillant-containing microsphere. In a typical radioimmunoassay, the microsphere is coated with a capture moiety, such as protein, A, or secondary antibodies, such as donkey-anti rabbit, sheep-anti-mouse antibodies. A sample, containing or suspected of containing the analyte (i.e. antigen) to be tested, is incubated in the presence of an antibody specific for that analyte, together with a quantity of a radiolabelled analyte. The antibody/analyte complex is captured by the secondary antibody and is detected by the emission of light. Any labelled antigen which remains unbound by the antibody, will be free in solution and be undetected. The assay therefore requires no separation step and the protocol has fewer pipetting steps compared with conventional, i.e. separation-based radioimmunoassays. It has been shown that in SPA-based assays, there is often an increase in assay precision and reproducibility, compared with traditional separation-based assays. Another advantage lies in the potential for increased sample throughput and capability for automation. (Cook, N. D., Drug Discovery Today (1996), 1, 287-294). The application of SPA to RIA methodology is not restricted to particular analytes or to types of molecule and in principle the technique can be applied in place of traditional separation-based assays. Some examples of RIAs developed using SPA are shown in Table 1.
TABLE 1Examples of Radioimmunoassays Developed using SPAAssayReferenceCyclic AMPHorton JK & Baxendale PM (1995), In:Methods in Molecular Biology, 41, pp. 91-105,Eds. Kendall, DA and Hill, SJ, Humana PressInc, Towota, NJCyclic GMPHeath R Bryant B & Horton JK (1992), In: TheBiology of Nitric Oxide. Part 2. Enzymology,Biochemistry and Immunology pp. 98-102, Eds.Moncada, S et al. Portland Press6-Keto-ProstoglandinBaxendale PM et al (1990) In: Advances inF1 alphaProstaglandins, Thromboxane and LeukotrieneResearch, 21, pp. 303-306, Eds. Samuelsson,B. et al, Raven PressAcyclovirTadepalli, SM, Topham, PA & Quinn, RP.(1990) Clin. Chem. 36, 1104Platelet ActivatingSugatani , J et al (1990), Life Sciences,Factor46, 1443-1450Abscisic AcidWhitford, PN. & Croker, SJ. (1991)Phytochemical Analysis, 2, 134-136AndrostenedioneFiet, J. et al (1991) Clin. Chem., 37,293RanitidineLinacre, P. & Morris, SE, (1992), In:Bioanalytical Approaches for Drugs, includinganti-asthmatics and metabolites, 22, pp.325-326 Eds. Reid, E. & Wilson, ID. Royal Societyof Chemistry, London
More recently, alternatives to scintillant-containing beads (fluomicrospheres) have been described for use in proximity assays. PCT Application No. WO 90/03844 (Wallac) discloses a microtitre well plate intended for binding assays. The sample plate is produced from a transparent scintillant-containing plastic by means of a vacuum thermoforming or injection moulding process. In principle, the walls of the microtitre well plate can be coated with a binding compound for the purpose of performing in vitro binding assays using radiolabelled reactants.
PCT Application No. WO 94/26413 discloses an apparatus and a method for studying cellular biochemical processes in real time. In one aspect, the application describes a multiwell plate, such as a microtitre well plate, in which the base of the plate is formed from a scintillant plastic material and the walls are formed from an opaque plastic material, the wells of the plate being adapted for the attachment or growth of cells. The scintillating microplates are designed for use in the real-time analysis of a wide spectrum of cell associated phenomena, and applications have been demonstrated in transport, cell motility, uptake, metabolism and other cell based processes. Cytostar™ scintillating microplates form the basis of a new technology introduced by Amersham International plc, for the study of cellular processes. In other applications, the scintillating microplates can be used for in vitro assays, for the measurement of ligands, analytes, etc. In this format a binding compound is bound to the walls of the microtitre well plate for reaction with label and analyte.
As an alternative to radioisotopic methods for performing immunoassays, non-radioactive systems have been introduced. Today, enzymes are the most widely used tracers. When in combination with colourimetric end-points, they provide highly sensitive, robust, precise, accurate and convenient immunoassays. A major breakthrough came with the introduction of ninety-six well microtitre plates. Inexpensive automatic colourimetric multiwell plate readers are available. A number of other non-isotopic labels have been described, of which luminescent and fluorescent labels are the most popular.
2. Cell Extraction Methods
Traditional methods for immunoassay depend on obtaining the samples in a sufficiently suitable state, i.e. sufficiently free from interfering factors. Usually this will involve a cellular extraction method. Numerous procedures are described which detail the extraction of intracellular molecules from cells. Typically these methods involve acid, solvent or solid phase methods to accomplish cell lysis and extraction of the molecule of interest. Methods for performing such extractions can be found in several publications. Further background information relating to cell extraction methods can be found in a review article by Goldberg & O'Toole. (Goldberg, N D & O'Toole, A G (1971); In: Methods of Biochemical Analysis, 20, Ed Glick D. pp 1-39 Interscience Publishers, Wiley, London)
Examples of cellular extraction methods are as follows.
2.1 Solvent Extraction (Horton & Baxendale, 1995; See Table 1 for Reference)
Ice-cold ethanol is added to cell cultures to give a final suspension volume of 65% (v/v) ethanol and the suspension allowed to settle. The supernatant is aspirated into test tubes, the remaining precipitate washed with ice-cold 65% (v/v) ethanol and the washings added to the appropriate tubes. The extracts are centrifuged at 2000 g for 15 minutes at 4° C. and the supernatant transferred to fresh tubes. The combined extract is then dried overnight, either under a stream of nitrogen at 60° C., in a vacuum oven for 8 hours, or in a centrifugal evaporator on a high temperature setting for 4 hours.
In this procedure, there is a possibility of overdrying, and this can result in difficulty in reconstituting the samples. The dried extracts are dissolved in a suitable volume of assay buffer before analysis.
2.2 Acid Extraction
Hancock et al, (J. of Receptor & Signal Transduction Research, 1995, 15, 557-579) describe an acid extraction method for intracellular molecules in which 0.2M hydrochloric acid is added to cells, and each separate sample is vortex mixed for 1-2 minutes. The sample is carefully neutralised to a pH that is compatible to the immunoassay, using stepwise addition of 10 μl aliquots of 2.5M sodium hydroxide, care being taken to measure the pH of the sample after each addition of alkali. This step is particularly critical, as the use of a non-optimal pH with an immunoassay can result in inaccurate measurement or non-measurement of analyte in the samples.
An alternative approach is described by Steiner, (In: Methods of Hormone Radioimmunoassay, 1979, Eds Jaffre, B M, & Behrman, H R pp. 3-17 Academic Press, New York), in which cell samples are homogenized in cold 6% (w/v) trichloroacetic acid at 4° C. to give a 10% (w/v) slurry. The sample is centrifuged at 2000 g for 15 minutes at 4° C. The supernatant is reserved and the pellet discarded. The supernatant is washed four times with five volumes of water-saturated diethyl ether, discarding the upper layer after each wash. The aqueous extract is lyophilized overnight or dried under a stream of nitrogen at 60° C. overnight and the dried extract dissolved in a suitable volume of assay buffer before analysis.
2.3 Solid Supports (e.g. Ion Exchange or ‘Amprep’ Columns)
A protocol for the extraction of intracellular molecules by ion exchange chromatography, using disposable minicolumns, has been described previously (Horton & Baxendale, 1995; see Table 1 for Reference). The columns (for example, ion exchange SAX columns) are used with a vacuum manifold and a vacuum pump. The columns are prepared by applying a vacuum and rinsing with 2 ml 100% methanol, followed by washing with 2 ml of water, taking careful precautions so as not to allow the solid support to dry, or to allow the flow rate to exceed 5 ml/minute. The cultured cells are applied directly to the column and washed with 3 ml 100% methanol. Three milliliters of acidified methanol (prepared by diluting concentrated hydrochloric acid to 0.1M with absolute methanol) is added to the column and the eluate collected. The fractions are dried using a stream of nitrogen or in a vacuum oven overnight (see above). The samples are reconstituted in assay buffer, as described above, before assay.
2.4 Detergent Methods
Other methods are known for the extraction of nucleic acid samples and nucleotides such as ATP. For example, Lundin and Anson (PCT WO 92/12253) describe a method for extracting an intracellular component in which bacterial cells are lysed with a detergent which is subsequently neutralised by addition of a cyclodextrin. A cellular component (e.g. ATP, DNA or RNA) liberated is subsequently measured or processed using biochemical or molecular biology (non-immunoassay) techniques such as the firefly luciferase and polymerase chain reaction assays. No reference is made in this patent application to one-step assays, homogeneous immunoassays, including scintillation proximity assay methods, or separation based immunoassay techniques.
EP 0 309 184 (Lumac) describes a method for the extraction of ATP from a microorganism with an ATP releasing agent and contacting the resultant solution with a neutralising agent which acts substantially to eliminate the distorting effect the releasing agent on the subsequent ATP assay. In EP 0 309 184 the releasing agent is preferably a cationic surface active agent which is preferably contacted with a non-ionic surface active neutralising agent.
The use of cyclodextrins to remove surfactants from solutions and surfaces and surfaces has been described previously in European Patent Application EP 301 847 (P. Khanna and R. Dworschack). According to this patent application, surfactants can be removed and cleaned from solutions and containers used in biochemical reactions by immobilised cyclodextrins. EP 286367 (Khanna et al) describes cyclodextrins as neutralisers of surfactants used as storage stabilisers for enzymes which are used as tracers in enzyme immunoassays. In a review, various applications of cyclodextrins in biological and chemical reactions have been described. (J. Szejtli, Cyclodextrins in Diagnostics, Kontakte [Darmstadt] 1988 [1]. 31-36).
The use of cyclodextrins, to neutralise surfactants added as extractants to release intracellular molecules, in a simple, single-step extraction and measurement immunoassay system has not been described previously.
All of the above prior art methods for immunoassays suffer from a number of disadvantages, including:    i) Unable to process large numbers of cells samples    ii) Time consuming    iii) Labour intensive    iv) Prone to errors because of the large number of steps    v) The need to remove the cell extraction reagent before further processing and measuring can take place. If this is not carried out, then accurate measurements may not take place, or, indeed, could result in total assay inhibition, and therefore measurement of the substance in the cellular extract is prevented.
In all of the traditional methods of sample preparation for radioimmunoassay, it has hitherto been necessary to perform separate lysis and extraction processes in order to obtain samples in a suitable form for subsequent measurement. The prior art methods therefore involve three separate processes which must be carried out sequentially, thereby adding to the time and cost of each immunochemical assay. In addition none of the prior art methods for the assay of intracellular components would be amenable to high throughput screening methods which are necessary if large numbers of samples are required to be processed. In this specification, data is presented whereby addition of a cellular lysis reagent to an immunoassay system, results in inhibition of antigen:antibody binding; and the inclusion of complex carbohydrates, such as cyclodextrins, restores the antigen:antibody binding event. In the preferred embodiment of the invention, 1% DTAB (dodecyl trimethyl ammonium bromide) is employed as a cellular lysis reagent (which inhibits antigen:antibody binding) and 2.5% alpha cyclodextrin is used as a sequestration reagent restoring antigen:antibody binding. These reagents, together with homogeneous immunoassay techniques, have enabled, for the first time, the establishment of a concerted one stage, single pot, cellular lysis and immunoassay system for the accurate measurement of intracellular molecules.
Thus, a novel, convenient and rapid method for the extraction and quantitation of target molecules is described here, which permits the growth of cells, the extraction of intracellular components and the subsequent assay of such components to be carried out in the same vessel. The technique is simple to perform and can be carried out with little technical intervention. Since few manipulations are necessary, the procedure is fully amenable to robotic automation